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1.  Clinical Implications of Power Toothbrushing on Fluoride Delivery: Effects on Biofilm Plaque Metabolism and Physiology 
Dental biofilms are implicated in the formation of caries and periodontal disease. A major constituent of the supragingival biofilm is Streptococcus mutans, which produces lactic acid from sucrose fermentation, enhancing enamel demineralization and eventual caries development. Caries prevention through F inhibits enamel demineralization and promotes remineralization. Fluoride also exerts effects on metabolic activities in the supragingival biofilm such as aerobic respiration, acid fermentation and dentrification. In experimental S. mutans biofilms, adding 1000 ppm F to an acidogenic biofilm resulting from 10% sucrose addition increased pH to pre-sucrose levels, suggesting inhibition of acid fermentation. F effects on metabolic activity and sucrose utilization in interproximal plaque biofilms were also recorded. Addition of 10% sucrose reduced pH from neutral to 4.2, but subsequent addition of 1000 ppm F increased pH by 1 unit, inhibiting acid fermentation. 10% Sucrose addition also stimulated denitrification, increasing production of nitrous oxide (N2O). Addition of 1000 ppm F suppressed denitrification, indicating an additional mechanism by which F exerts effects in the active interproximal biofilm. Finally, fluid dynamic activity by power tooth brushing enhanced F delivery and retention in an experimental S. mutans biofilm, suggesting a potential novel benefit for this intervention beyond mechanical plaque removal.
doi:10.1155/2010/651869
PMCID: PMC2855952  PMID: 20414341
2.  Fiber optic backscatter spectroscopic sensor to monitor enamel demineralization and remineralization in vitro 
In this study, a Fiber Optic Backscatter Spectroscopic Sensor (FOBSS) is used to monitor demineralization and remineralization induced changes in the enamel. A bifurcated fiber optic backscatter probe connected to a visible light source and a high resolution spectrophotometer was used to acquire the backscatter light spectrum from the tooth surface. The experiments were conducted in two parts. In Part 1, experiments were carried out using fiber optic backscatter spectroscopy on (1) sound enamel and dentine sections and (2) sound tooth specimens subjected to demineralization and remineralization. In Part 2, polarization microscopy was conducted to examine the depth of demineralization in tooth specimens. The enamel and dentine specimens from the Part-1 experiments showed distinct backscatter spectra. The spectrum obtained from the enamel-dentine combination and the spectrum generated from the average of the enamel and dentine spectral values were closely similar and showed characteristics of dentine. The experiments in Part 2 showed that demineralization and remineralization processes induced a linear decrease and linear increase in the backscatter light intensity respectively. A negative correlation between the decrease in the backscatter light intensity during demineralization and the depth of demineralization determined using the polarization microscopy was calculated to be p = -0.994. This in vitro experiment highlights the potential benefit of using FOBSS to detect demineralization and remineralization of enamel.
doi:10.4103/0972-0707.44053
PMCID: PMC2813093  PMID: 20142887
Fiber optic; demineralization; dentine; remineralization
3.  Nondestructive Assessment of Early Tooth Demineralization Using Cross-Polarization Optical Coherence Tomography 
New methods are needed for the nondestructive measurement of tooth demineralization and remineralization to monitor the progression of incipient caries lesions (tooth decay) for effective nonsurgical intervention and to evaluate the performance of anticaries treatments such as chemical treatments or laser irradiation. Studies have shown that optical coherence tomography (OCT) has great potential to fulfill this role since it can be used to measure the depth and severity of early lesions with an axial resolution exceeding 10 µm, it is easy to apply in vivo and it can be used to image the convoluted topography of tooth occlusal surfaces. In this paper, a review of the use of polarization-sensitive-OCT for the measurement of tooth demineralization is provided along with some recent results regarding improved methods for the detection of caries lesions in the earliest stages of development. Automated methods of analysis were used to measure the depth and severity of demineralized bovine enamel produced using simulated caries models that emulate demineralization in the mouth. Significant differences in the depth and integrated reflectivity from the lesions were detected after only a few hours of demineralization. These results demonstrate that cross-polarization-OCT is ideally suited for the nondestructive assessment of early demineralization.
doi:10.1109/JSTQE.2009.2033610
PMCID: PMC3109909  PMID: 21660217
Dental caries; polarization-sensitive-optical coherence tomography (PS-OCT); tooth demineralization
4.  Imaging early demineralization with PS-OCT 
New methods are needed for the nondestructive measurement of tooth demineralization and remineralization to monitor the progression of incipient caries lesions (tooth decay) for effective nonsurgical intervention and to evaluate the performance of anti-caries treatments such as chemical treatments or laser irradiation. Studies have shown that optical coherence tomography (OCT) has great potential to fulfill this role since it can be used to measure the depth and severity of early lesions with an axial resolution exceeding 10-μm, it is easy to apply in vivo and it can be used to image the convoluted topography of tooth occlusal surfaces. In this paper we attempt to determine the earliest stage at which we can detect significant differences in lesion severity. Automated methods of analysis were used to measure the depth and severity of demineralized bovine enamel produced using a simulated caries model that emulates demineralization in the mouth. Significant differences in the depth and integrated reflectivity from the lesions were detected after only a few hours of demineralization. These results demonstrate that cross polarization OCT is ideally suited for the nondestructive assessment of early demineralization.
doi:10.1117/12.849343
PMCID: PMC3293401  PMID: 22399835
polarization; optical coherence tomography; tooth demineralization; dental caries
5.  Atomic force microscopic comparison of remineralization with casein-phosphopeptide amorphous calcium phosphate paste, acidulated phosphate fluoride gel and iron supplement in primary and permanent teeth: An in-vitro study 
Context:
Demineralization of tooth by erosion is caused by frequent contact between the tooth surface and acids present in soft drinks.
Aim:
The present study objective was to evaluate the remineralization potential of casein-phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste, 1.23% acidulated phosphate fluoride (APF) gel and iron supplement on dental erosion by soft drinks in human primary and permanent enamel using atomic force microscopy (AFM).
Materials and Methods:
Specimens were made from extracted 15 primary and 15 permanent teeth which were randomly divided into three treatment groups: CPP-ACP paste, APF gel and iron supplement. AFM was used for baseline readings followed by demineralization and remineralization cycle.
Results and Statistics:
Almost all group of samples showed remineralization that is a reduction in surface roughness which was higher with CPP-ACP paste. Statistical analysis was performed using by one-way ANOVA and Mann-Whitney U-test with P < 0.05.
Conclusions:
It can be concluded that the application of CPP-ACP paste is effective on preventing dental erosion from soft drinks.
doi:10.4103/0976-237X.128672
PMCID: PMC4012123  PMID: 24808700
Atomic force microscope; dental erosion; remineralization
6.  Methods for Monitoring Erosion Using Optical Coherence Tomography 
Since optical coherence tomography is well suited for measuring small dimensional changes on tooth surfaces it has great potential for monitoring tooth erosion. The purpose of this study was to explore different approaches for monitoring the erosion of enamel. Application of an acid resistant varnish to protect the tooth surface from erosion has proven effective for providing a reference surface for in vitro studies but has limited potential for in vivo studies. Two approaches which can potentially be used in vivo were investigated. The first approach is to measure the remaining enamel thickness, namely the distance from the tooth surface to the dentinal-enamel junction (DEJ). The second more novel approach is to irradiate the surface with a carbon dioxide laser to create a reference layer which resists erosion. Measuring the remaining enamel thickness proved challenging since the surface roughening and subsurface demineralization that commonly occurs during the erosion process can prevent resolution of the underlying DEJ. The areas irradiated by the laser manifested lower rates of erosion compared to the non-irradiated areas and this method appears promising but it is highly dependent on the severity of the acid challenge.
doi:10.1117/12.2011013
PMCID: PMC3898817  PMID: 24465117
erosion; optical coherence tomography; caries prevention; carbon dioxide laser
7.  Clinical Monitoring of Early Caries Lesions using Cross Polarization Optical Coherence Tomography 
New methods are needed for the nondestructive measurement of tooth demineralization and remineralization and to monitor the progression of incipient caries lesions (tooth decay) for effective nonsurgical intervention and to evaluate the performance of anti-caries treatments such as chemical treatments or laser irradiation. Studies have shown that optical coherence tomography (OCT) has great potential to fulfill this role, since it can be used to measure the depth and severity of early lesions with an axial resolution exceeding 10-μm. It is easy to apply in vivo and it can be used to image the convoluted topography of tooth occlusal surfaces. In this paper we present early results from two clinical studies underway to measure the effect of fluoride intervention on early lesions. CP-OCT was used to monitor early lesions on enamel and root surfaces before and after intervention with fluoride varnish. The lesion depth and internal structure were resolved for all the lesions examined and some lesions had well defined surface zones of lower reflectivity that may be indicative of arrested lesions. Changes were also noted in the structure of some of the lesions after fluoride intervention.
doi:10.1117/12.2011014
PMCID: PMC3864974  PMID: 24353384
cross polarization optical coherence tomography; tooth demineralization; dental caries; root caries
8.  Design and Characterization of an Acid-Activated Antimicrobial Peptide 
Chemical biology & drug design  2009;75(1):127-132.
Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/ remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals creation of an acidic environment favors growth of acid enduring and acid generating species, which causes further reduction in the plaque pH. In this study we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype “acid-activated peptides” (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries.
doi:10.1111/j.1747-0285.2009.00904.x
PMCID: PMC2790279  PMID: 19878192
Targeted antimicrobial therapy; pH dependent antimicrobial activity; biofilm; Streptococcus mutans
9.  Imaging in vivo secondary caries and ex vivo dental biofilms using cross-polarization optical coherence tomography 
Dental Materials  2012;28(7):792-800.
Objectives
Conventional diagnostic methods frequently detect only late stage enamel demineralization under composite resin restorations. The objective of this study is to examine the subsurface tooth-composite interface and to assess for the presence of secondary caries in pediatric patients using a novel Optical Coherence Tomography System with an intraoral probe.
Methods
A newly designed intraoral cross polarization swept source optical coherence tomography (CP-OCT) imaging system was used to examine the integrity of the enamel-composite interfaces in vivo. Twenty two pediatric subjects were recruited with either recently placed or long standing composite restorations in their primary teeth. To better understand how bacterial biofilms cause demineralization at the interface, we also used the intraoral CP-OCT system to assess ex vivo bacterial biofilm growth on dental composites.
Results
As a positive control, cavitated secondary carious interfaces showed a 18.2 dB increase (p<0.001), or over 1-2 orders of magnitude higher, scattering than interfaces associated with recently placed composite restorations. Several long standing composite restorations, which appeared clinically sound, had a marked increase in scattering than recently placed restorations. This suggests the ability of CP-OCT to assess interfacial degradation such as early secondary caries prior to cavitation. CP-OCT was also able to image ex vivo biofilms on dental composites and assess their thickness.
Significance
This paper shows that CP-OCT imaging using a beam splitter based design can examine the subsurface interface of dental composites in human subjects. Furthermore, the probe dimensions and acquisition speed of the CP-OCT system allowed for analysis of caries development in children.
doi:10.1016/j.dental.2012.04.004
PMCID: PMC3372533  PMID: 22578989
10.  Effects of easy-to-perform procedures to reduce bacterial colonization with Streptococcus mutans and Staphylococcus aureus on toothbrushes 
It is well known that dental caries and periodontitis are the consequence of bacterial colonization and biofilm formation on the enamel surface. The continuous presence of bacterial biofilms on the tooth surface results in demineralization of the tooth enamel and induces an inflammatory reaction of the surrounding gums (gingivitis). The retention and survival of microorganisms on toothbrushes pose a threat of recontamination especially for certain patients at risk for systemic infections originating from the oral cavity, e.g., after T-cell depleted bone marrow transplantation. Thus, the effects of different decolonization schemes on bacterial colonization of toothbrushes were analyzed, in order to demonstrate their applicability to reduce the likelihood of (auto-)reinfections.
Toothbrushes were intentionally contaminated with standardized suspensions of Streptococcus mutans or Staphylococcus aureus. Afterwards, the toothbrushes were exposed to rinsing under distilled water, rinsing and drying for 24 h, 0.2% chlorhexidine-based decolonization, or ultraviolet (UV) radiation. The remaining colony forming units were compared with freshly contaminated positive controls. Each experiment was nine-fold repeated. Bi-factorial variance analysis was performed; significance was accepted at P < 0.05.
All tested procedures led to a significant reduction of bacteral colonization irrespective of the toothbrush model, the brush head type, or the acitivity state. Chlorhexidine-based decolonization was shown to be superior to rinsing and slightly superior to rinsing and drying for 24 h, while UV radiation was similarly effective as chlorhexidine. UV radiation was slightly less prone to species-dependent limitations of its decolonizing effects by bristle thickness of toothbrushes than chlorhexidin.
Reduction of bacterial colonization of toothbrushes might reduce the risk of maintaining bacterial infections of the upper respiratory tract. Accordingly, respective procedures are advisable, particularly as they are cheap and easy to perform.
doi:10.1556/EuJMI.3.2013.3.9
PMCID: PMC3832098  PMID: 24265940
bacterial colonization; decolonization; oral hygiene; Staphylococcus aureus; Streptococcus mutans; toothbrush
11.  Structural Identities of Four Glycosylated Lipids in the Oral Bacterium Streptococcus mutans UA159 
Biochimica et biophysica acta  2013;1831(7):1239-1249.
The cariogenic bacterium Streptococcus mutans is an important dental pathogen that forms biofilms on tooth surfaces, which provide a protective niche for the bacterium where it secretes organic acids leading to the demineralization of tooth enamel. Lipids, especially glycolipids are likely to be key components of these biofilm matrices. The UA159 strain of S. mutans was among the earliest microorganisms to have its genome sequenced. While the lipids of other S. mutans strains have been identified and characterized, lipid analyses of UA159 have been limited to a few studies on its fatty acids. Here we report the structures of the four major glycolipids from stationary-phase S. mutans UA159 cells grown in standing cultures. These were shown to be monoglucosyldiacylglycerol (MGDAG), diglucosyldiacylglycerol (DGDAG), diglucosylmonoacylglycerol (DGMAG) and, glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). The structures were determined by high performance thin-layer chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. The glycolipids were identified by accurate, high resolution, and tandem mass spectrometry. The identities of the sugar units in the glycolipids were determined by a novel and highly efficient NMR method. All sugars were shown to have α-glycosidic linkages and DGMAG was shown to be acylated in the sn-1 position by NMR. This is the first observation of unsubstituted DGMAG in any organism and the first mass spectrometry data for GPDGDAG.
doi:10.1016/j.bbalip.2013.03.012
PMCID: PMC3672342  PMID: 23562838
Bacterium; glycolipids; lipidomics; dental pathogen
12.  Effect of an Orphan Response Regulator on Streptococcus mutans Sucrose-Dependent Adherence and Cariogenesis  
Infection and Immunity  2003;71(8):4351-4360.
Streptococcus mutans is the principal acidogenic component of dental plaque that demineralizes tooth enamel, leading to dental decay. Cell-associated glucosyltransferases catalyze the sucrose-dependent synthesis of sticky glucan polymers that, together with glucan binding proteins, promote S. mutans adherence to teeth and cell aggregation. We generated an S. mutans Tn916 transposon mutant, GMS315, which is defective in sucrose-dependent adherence and significantly less cariogenic than the UA130 wild-type progenitor in germfree rats. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and N-terminal sequence analysis confirmed the absence of a 155-kDa glucosyltransferase S (Gtf-S) from GMS315 protein profiles. Mapping of the unique transposon insertion in GMS315 revealed disruption of a putative regulatory region located upstream of gcrR, a gene previously described by Sato et al. that shares significant amino acid identity with other bacterial response regulators (Y. Sato, Y. Yamamoto, and H. Kizaki, FEMS Microbiol. Lett. 186: 187-191, 2000). The gcrR regulator, which we call “tarC,” does not align with any of the 13 proposed two-component signal transduction systems derived from in silico analysis of the S. mutans genome, but rather represents one of several orphan response regulators in the genome. The results of Northern hybridization and/or real-time reverse transcription-PCR experiments reveal increased expression of both Gtf-S and glucan binding protein C (GbpC) in a tarC knockout mutant (GMS900), thereby supporting the notion that TarC acts as a negative transcriptional regulator. In addition, we noted that GMS900 has altered biofilm architecture relative to the wild type and is hypocariogenic in germfree rats. Taken collectively, these findings support a role for signal transduction in S. mutans sucrose-dependent adherence and aggregation and implicate TarC as a potential target for controlling S. mutans-induced cariogenesis.
doi:10.1128/IAI.71.8.4351-4360.2003
PMCID: PMC166011  PMID: 12874312
13.  Elevated antibody to D-alanyl lipoteichoic acid indicates caries experience associated with fluoride and gingival health 
BMC Oral Health  2002;2:2.
Background
Acidogenic, acid-tolerant bacteria induce dental caries and require D-alanyl glycerol lipoteichoic acid (D-alanyl LTA) on their cell surface. Because fluoride inhibits acid-mediated enamel demineralization, an elevated antibody response to D-alanyl LTA may indicate subjects with more acidogenic bacteria and, therefore, an association of DMFT with fluoride exposure and gingival health not apparent in low responders.
Methods
Cluster analysis was used to identify low antibody content. Within low and high responders (control and test subjects), the number of teeth that were decayed missing and filled (DMFT), or decayed only (DT) were regressed against fluoride exposure in the water supply and from dentrifice use. The latter was determined from gingival health: prevalences of plaque (PL) and bleeding on probing (BOP), and mean pocket depth (PD). Age was measured as a possible confounding cofactor.
Results
In 35 high responders, DMFT associated with length of exposure to fluoridated water (F score), PL and BOP (R2 = 0.51, p < 0.001), whereas in 67 low D-ala-IgG responders, DMFT associated with PL, age, and PD (R2 = 0.26, p < 0.001). BOP correlated strongly with number of 7 7 decayed teeth (DT) in 54 high responders (R2 = 0.57, p < 0.001), but poorly in 97 low responders (R2 = 0.12, p < 0.001). The strength of the PD association with DMFT, or of BOP with DT, in high responders significantly differed from that in low responders (p < 0.05).
Conclusion
Caries associates with gingival health and fluoridated water exposure in high D-alanyl LTA antibody responders.
doi:10.1186/1472-6831-2-2
PMCID: PMC100323  PMID: 11922867
14.  Imaging laser irradiated enamel surfaces with polarization sensitive optical coherence tomography 
Several studies have shown that lasers can be used to modify the surface morphology and chemical composition of tooth enamel to render it less soluble. Other studies have shown that Polarization Sensitive Optical Coherence Tomography (PS-OCT) can be used to non-destructively measure the efficacy of fluoride in inhibiting the development of artificial caries lesions. The purpose of this study was to determine if PS-OCT can be used to measure inhibition of enamel demineralization after CO2 laser irradiation. Polarized light microscopy and microradiography were used to measure lesion severity on histological thin sections for comparison. PS-OCT was able to measure a significant reduction in the integrated reflectivity due to inhibition by the laser even though the laser modification of the enamel surface caused a slight increase in reflectivity. This study shows that the PS-OCT is well-suited for in vivo measurements of caries inhibition after laser treatments.
doi:10.1117/12.778789
PMCID: PMC3168543  PMID: 21909224
15.  Shedding New Light on Early Caries Detection 
Dental caries continues to be a common chronic disease among various population groups. Patient care can be improved with detection at the earliest stage. However, current techniques do not have sufficient sensitivity and specificity. We discuss 2 new methods — optical coherence tomography (OCT) and polarized Raman spectroscopy (PRS) — that are potentially useful for early caries detection and monitoring.
OCT produces morphologic depth images of near-surface tissue structures with a resolution that is an order of magnitude greater than ultrasound imaging. Based on measurement of back-scattered near infrared light, OCT shows that sound enamel causes high-intensity back-scattering at the tooth surface that decreases rapidly with depth. In contrast, incipient lesions cause higher light back-scattering at the tooth surface and subsurface scattering indicative of porosity caused by demineralization. The scatter region within the enamel correlates well with the classical triangular shape of subsurface lesions observed in histologic sections. OCT imaging not only allows identification of incipient lesions, but also provides information on surface integrity and lesion depth.
PRS furnishes biochemical information about the tooth's composition, mineral content and crystallinity. The depolarization ratio derived from the dominant phosphate peak of hydroxyapatite in sound teeth is consistently lower than that from incipient caries. This difference is attributed to the change in enamel crystallite morphology or orientation that occurs with acid demineralization. Thus, PRS can be used to confirm suspect lesions determined by OCT and rule out false-positive signals from non-carious anomalies.
The combination of OCT and PRS provides a new detection method with high sensitivity and specificity that will improve caries management and patient care. Future studies are aimed at developing intraoral probes to validate the findings in vivo.
PMCID: PMC2700544  PMID: 19126361
16.  Effect of Fluoride on Artificial Caries Lesion Progression and Repair in Human Enamel: Regulation of Mineral Deposition and Dissolution under In Vivo‐Like Conditions 
Archives of oral biology  2006;52(2):110-120.
Objective
This study was carried out to determine in vitro the effect of fluoride on 1) the demineralization of sound human enamel and 2) the progression of artificial caries‐like lesions, under relevant oral conditions.
Methods
Thin sections of sound human enamel were exposed to solutions undersaturated with respect to tooth enamel to a degree similar to that found in dental plaque fluid following sucrose exposure in vivo, containing fluoride concentrations (0 – 0.38 ppm) found in plaque fluid. Mineral changes were monitored for 98 days, using quantitative microradiography. The effect of fluoride (1.0 – 25.0 ppm) on the progression of artificial caries‐like lesions was similarly studied.
Results
Fluoride concentrations of 0.19 ppm and greater were found to prevent the demineralization of sound enamel in vitro. However, significantly higher concentrations of fluoride (25.0 ppm) were required to prevent further demineralization of artificial caries‐like lesions. Demineralizing solutions with intermediate fluoride concentrations (2.1 – 10.1 ppm) induced simultaneously remineralization in the outer portion of the lesion and demineralization in the inner portion. Simultaneous remineralization and demineralization were also observed in hydroxyapatite pellets.
Conclusions
Our results show that the observed effect of fluoride on enamel demineralization is not solely a function of bulk solution properties, but also depends on the caries‐status of the enamel surface. A mechanistic model presented indicates that, in comparison to sound enamel surfaces, higher concentrations of fluoride are required to prevent the progression of artificial caries‐like lesions under in vivo‐like conditions since the diffusion of mineral ions that promote remineralization is rate‐limiting.
doi:10.1016/j.archoralbio.2006.08.012
PMCID: PMC2040327  PMID: 17049334
dental enamel; remineralization; demineralization; mineral distribution; fluoride
17.  Imaging Early Demineralization on Tooth Occlusal Surfaces with a High Definition InGaAs Camera 
In vivo and in vitro studies have shown that high contrast images of tooth demineralization can be acquired in the near-IR due to the high transparency of dental enamel. The purpose of this study is to compare the lesion contrast in reflectance at near-IR wavelengths coincident with high water absorption with those in the visible, the near-IR at 1300-nm and with fluorescence measurements for early lesions in occlusal surfaces. Twenty-four human molars were used in this in vitro study. Teeth were painted with an acid-resistant varnish, leaving a 4×4 mm window in the occlusal surface of each tooth exposed for demineralization. Artificial lesions were produced in the exposed windows after 1 & 2-day exposure to a demineralizing solution at pH 4.5. Lesions were imaged using NIR reflectance at 3 wavelengths, 1310, 1460 and 1600-nm using a high definition InGaAs camera. Visible light reflectance, and fluorescence with 405-nm excitation and detection at wavelengths greater than 500-nm were also used to acquire images for comparison. Crossed polarizers were used for reflectance measurements to reduce interference from specular reflectance. The contrast of both the 24 hr and 48 hr lesions were significantly higher (P<0.05) for NIR reflectance imaging at 1460-nm and 1600-nm than it was for NIR reflectance imaging at 1300-nm, visible reflectance imaging, and fluorescence. The results of this study suggest that NIR reflectance measurements at longer near-IR wavelengths coincident with higher water absorption are better suited for imaging early caries lesions.
doi:10.1117/12.2011015
PMCID: PMC3865214  PMID: 24357911
Near-IR imaging; enamel; demineralization; dental caries; polarization; artificial lesions
18.  Effects of Ion-Releasing Tooth-Coating Material on Demineralization of Bovine Tooth Enamel 
We compared the effect of a novel ion-releasing tooth-coating material that contained S-PRG (surface-reaction type prereacted glass-ionomer) filler to that of non-S-PRG filler and nail varnish on the demineralization of bovine enamel subsurface lesions. The demineralization process of bovine enamel was examined using quantitative light-induced fluorescence (QLF) and electron probe microanalyzer (EPMA) measurement. Ion concentrations in demineralizing solution were measured using inductively coupled plasma atomic (ICP) emission spectrometry and an ion electrode. The nail varnish group and the non-S-PRG filler group showed linear demineralization. Although the nail varnish group and the non-S-PRG filler group showed linear demineralization, the S-PRG filler group did not. Further, plane-scanning by EPMA analysis in the S-PRG filler group showed no changes in Ca ion distribution, and F ions showed peak levels on the surface of enamel specimens. Most ions in the demineralizing solution were present at higher concentrations in the S-PRG filler group than in the other two groups. In conclusion, only the S-PRG filler-containing tooth-coating material released ions and inhibited demineralization around the coating.
doi:10.1155/2014/463149
PMCID: PMC3918696  PMID: 24578706
19.  Polarization Sensitive Optical Coherence Tomographic Imaging of Artificial Demineralization on Exposed Surfaces of Tooth Roots 
Background and Objectives
The purpose of this study was to assess the potential of polarization sensitive optical coherence tomography (PS-OCT) to non-destructively measure the depth and severity of artificial demineralization on exposed root surfaces and measure the degree of inhibition by topical fluoride. Although PS-OCT imaging studies have demonstrated the utility of PS-OCT for imaging carious lesions on enamel and dentin surfaces the influence of the cementum layer that is present on intact root surfaces has not been investigated.
Materials and Methods
In this study, extracted human tooth roots were partitioned into three sections with one partition treated with topical fluoride, one partition protected from demineralization with acid resistant varnish, and one partition exposed to a demineralization solution, producing artificial lesions approximately 200-µm deep in root dentin. The lesion depth, remaining cementum thickness and the integrated reflectivity for lesion areas was measured with PS-OCT. These measurements were also compared with more established methods of measuring demineralization, namely transverse microradiography (TMR) and polarized light microscopy (PLM)
Results
PS-OCT was able to measure a significant increase in the reflectivity between lesion areas and sound root surfaces. In contrast to dentin, the cementum layer manifests minimal reflectivity in the PS-OCT images allowing nondestructive measurement of the remaining cementum thickness. The reflectivity of the cementum layer did not increase significantly after substantial demineralization, however it did manifest considerable shrinkage in a fashion similar to dentin and that shrinkage could be measured with OCT.
Significance
This study demonstrates that PS-OCT can be used to measure demineralization non-destructively on root surfaces and assess inhibition of demineralization by anticaries agents.
doi:10.1016/j.dental.2008.11.014
PMCID: PMC2701248  PMID: 19167052
optical coherence tomography; polarization; root dentin; cementum; artificial lesions; caries inhibition; microradiography; topical fluoride
20.  Analysis of Streptococcus mutans Proteins Modulated by Culture under Acidic Conditions 
Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.
doi:10.1128/AEM.68.5.2382-2390.2002
PMCID: PMC127557  PMID: 11976112
21.  Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms 
BMC Microbiology  2011;11:14.
Background
The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella.
Results
As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers.
Conclusions
Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.
doi:10.1186/1471-2180-11-14
PMCID: PMC3032641  PMID: 21247450
22.  Near-IR Polarization Imaging of Sound and Carious Dental Enamel 
A thorough understanding of how polarized near-IR light propagates through sound and carious dental hard tissues is important for the development of dental optical imaging systems. New optical imaging tools for the detection and assessment of dental caries (dental decay) such as near-IR imaging and optical coherence tomography can exploit the enhanced contrast provided by polarization sensitivity. In this investigation, an automated system was developed to collect images for the full 16-element Mueller Matrix. The polarized light was controlled by linear polarizers and liquid crystal retarders and the 36 images were acquired as the polarized near-IR light propagates through the enamel of extracted human thin tooth sections. In previous work, we reported that polarized light is rapidly depolarized by demineralized enamel, and sound and demineralized dentin.1 The rapid depolarization of polarized light by dental caries in the near-IR provides high contrast for caries imaging and detection. In this initial study, major differences in the Mueller matrix elements were observed in both sound and demineralized enamel which supports this approach and warrants further investigation.
doi:10.1117/12.849341
PMCID: PMC3251263  PMID: 22228980
Mueller-Matrix; polarization; Imaging; Near-IR; dental hard tissue
23.  An Automated Digital Microradiography System for Assessing Tooth Demineralization 
Digital Transverse microradiography (TMR) offers several advantages over film based methods including real-time image acquisition, excellent linearity with exposure, and it does not require expensive specialized film. The purpose of this work was to demonstrate that a high-resolution digital microradiography system can be used to measure the volume percent mineral loss for sound and demineralized enamel and dentin thin sections from 150–350-μm in thickness. A custom fabricated digital microradiography system with ~ 2-μm spatial resolution consisting of a digital x-ray imaging camera, a computerized high-speed motion control system and a high-intensity copper Kα; x-ray source was used to determine the volume percent mineral content of sound and demineralized tooth sections. The volume percent mineral loss was compared with cross-sectional microhardness measurements on sound extracted human teeth. The correlation between microhardness and microradiography was excellent (Pr=0.99) for section thickness ranging from 59–319-μm (n=13). The attenuation was linear with varying exposure time from 1–10 seconds. Digital TMR is an effective and rapid method for the assessment of the mineral content of enamel and dentin thin sections.
doi:10.1117/12.816868
PMCID: PMC3175372  PMID: 21935288
dental enamel; dental caries; microradiography
24.  Methods for calculating the severity of demineralization on tooth surfaces from PS-OCT scans 
Several studies have demonstrated that polarization sensitive optical coherence tomography (PS-OCT) can be used to nondestructively measure the severity of subsurface demineralization in enamel and dentin. The reflectivity in the polarization state orthogonal to the initial linear polarization incident on the tissue is low at sound tissues interfaces and high in demineralized areas that strongly scatter and depolarize the light. The purpose of this study was to develop improved algorithms for assessing the depth and severity of demineralization from PS-OCT scans for use with 2D and 3D tomographic images. Subsurface caries-like lesions of increasing depth and severity were produced in adjoining windows on ten bovine enamel samples by exposure to demineralization over periods of 1 to 4 days. Each sample also had a sound window to be used as a control. PS-OCT scans were acquired for each sample and analyzed using various methods to calculate the lesion depth and area. Algorithms were developed and used to automatically detect the lesion depth and area, and calculate the volume for improved assessment of lesion severity. Both fixed-depth and automatic edge-finding algorithms were able to detect significant differences between each of the days and sound enamel. The lesion depth and mineral loss were also measured with polarized light microscopy and transverse microradiography after sectioning the teeth. Mean lesion depths ranged from 40 to 100 μm. This demonstrates the edge-finding algorithm can be used to automatically determine the depth and severity of early lesions for the rapid analysis of PS-OCT images.
doi:10.1117/12.816867
PMCID: PMC3175369  PMID: 21935290
Enamel; caries; PS-OCT; image processing algorithms; early demineralization; edge detection
25.  High Contrast Near-infrared Polarized Reflectance Images of Demineralization on Tooth Buccal and Occlusal Surfaces at λ=1310-nm 
Lasers in surgery and medicine  2009;41(3):208-213.
Background and Objectives:
Sound enamel manifests peak transparency in the near-IR (NIR) at 1310-nm, therefore the near-IR is ideally suited for high contrast imaging of dental caries. The purpose of this study was to acquire images of early demineralized enamel on the buccal and occlusal surfaces of extracted human teeth using NIR reflectance imaging and compare the contrast of those images with the contrast of images taken using other methods.
Materials and Methods:
Fifteen human molars were used in this in vitro study. Teeth were painted with a clear acid-resistant varnish, leaving two 2×2 mm windows on the buccal and occlusal surfaces of each tooth for demineralization. Artificial lesions were produced in the exposed windows after a 2-day exposure to a demineralizing solution at pH-4.5. Lesions were imaged using NIR transillumination, NIR and visible light reflectance, and fluorescence imaging methods. Crossed polarizers were used where appropriate to improve contrast. Polarization sensitive optical coherence tomography (PS-OCT) was also used to non-destructively assess the depth and severity of demineralization in each sample window.
Results:
NIR reflectance imaging had the highest image contrast for both the buccal and occlusal groups and it was significantly higher contrast than visible light reflectance (P<0.05).
Conclusion:
The results of the study suggest that NIR reflectance imaging is a promising new method for acquiring high contrast images of early demineralization on tooth surfaces.
doi:10.1002/lsm.20746
PMCID: PMC2689647  PMID: 19291753
demineralization; dental caries; enamel; near-IR Imaging; polarization

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